Compared to plant biomass currently used in many areas, algae have a lower content of recalcitrant components, such as lignin to use. Therefore, algae are advantageous in that they can be converted much more easily into a monosaccharide which is a substrate for producing bioenergy and biochemicals. Also, by not using food resources, algae are free from the problem of using food resources in producing energy. For these reasons, algae are receiving attention as important biomass in an aspect of the production of biochemicals including alternative energy.
Among algae, especially red algae (e.g., Gelidium amansii) is not only a raw material for the production of such bioenergy and biochemicals, but is also reported to contain agarooligosaccharides, which exhibit excellent physiological activity such as antioxidation, anti-inflammation, anti-cancer, anti-allergy, whitening, and moisturization, as a component, such that it is usefully employed in pharmaceutical and cosmetic fields (U.S. Pat. No. 7,662,2291 by Tomono et al. (2009); U.S. Patent No. 69/143,282 by Enoki et al. (2005)).
As a main constituent, red algae contain agarose which is a polymer having neoagarobiose (i.e. 3,6-anhydro-L-galactose and D-galactose bonded by an α-1,3-linkage) as a basic unit that is connected with another unit by a β-1,4-linkage. The present inventors empirically proved the fact that the physiological functionality of 3,6-anhydro-L-galactose (Yun et at (2013) Appl Microbiol Biotechnol. 97(7):2961-70). Also reported in relation to the production of the 3,6-anhydro-L-galactose is a preparation process of 3,6-anhydro-L-galactose and D-galactose by a mild chemical pretreatment and enzymatic saccharification to replace chemical treatment methods (Jol et at (1999) Anal Biochem. 268, 213-222; Kim et at (2010) Bull Korean Soc. 31(2) 511-514) that have problems of a very low yield and a high possibility of excessive hydrolysis.
Agarose is depolymerized by an exo-type Aga50D enzyme after a chemical pretreatment into neoagarobiose and agarotriose (D-galactose-β-1,4 linkage-3,6-anhydro-L-galactose-α-1,3 linkage-D-galactose) which are main reaction products. Then, the neoagarobiose is ultimately hydrolyzed into 3,6-anhydro-L-galactose and D-galactose by SdNABH (Korean Patent No. 10-1293668), which is an α-neoagarobiose hydrolase. During this process, a chemical pretreatment under mild conditions is essential to improve the reactivity of the Aga50D enzyme. However, agarotriose produced using a chemically pretreated substrate cannot be ultimately decomposed into 3,6-anhydro-L-galactose and D-galactose and remains as a reaction residue.
If the remaining agarotriose could be ultimately decomposed into 3,6-anhydro-L-galactose and D-galactose, a larger improvement in the production yield of 3,6-anhydro-L-galactose and D-galactose would be expected. Since endo-type and exo-type β-agarases cannot hydrolyze a substrate such as agarotriose, another approach of decomposing agarotriose into D-galactose and neoagarobiose using a β-galactosidase was tried, but commercially available β-galactosidases did not show hydrolytic activity for agarooligosaccharides.